Many eukaryotic cells undergo chemotaxis, directed movement towards a soluble ligand. This process is necessary for many biological functions, including wound healing, axonal guidance, and the aggregation of Dictyostelium cells. Chemotaxis also plays a role in disease states such as arthritis, cancer, and multiple sclerosis. Directional cell movement requires a defined cell polarity in which components of the cytoskeleton are differentially localized at the leading edge of a migrating cell as well as its retracting posterior. This polarization can be initiated by the chemoattractant binding of heterotrimeric G protein-coupled membrane- bound receptors, and subsequent activation of downstream signal transduction pathways direct reorganization of the actin and myosin cytoskeleton. Our previous studies suggested that a polarized actin cytoskeleton is achieved by concentrating regulators of actin assembly such as WASP and WIP in subdomains at the cell cortex. The goal of this study is to understand how the subcellular localization and activation of WASP is regulated during chemotaxis. We hypothesize that a complex interplay between PCH family proteins, CLP1/2, and RacC leads to polarized trafficking of WASP and ACA, resulting in cell polarity establishment via the polarized organization of actin cytoskeleton and asymmetric distribution of ACA during Dictyostelium chemotaxis. To test this hypothesis, we propose the following specific aims: 1) To elucidate the role of PCH family proteins, CLP1/2 and RacC in controlling function and localization of WASP during vesicle formation. 2) To investigate roles of CLP1/2, RacC, and Unc104 in the trafficking of WASP- and ACA-vesicles. 3) To study how polarized trafficking of WASP- and ACA-vesicles contribute to establish cell polarity during chemotaxis. It is anticipated that these studies will serve not only to provide new insights on how exocytic vesicle formation from Golgi is controlled, but also to understand how cellular polarity can be achieved b vesicle trafficking during chemotaxis. PUBLIC HEALTH RELEVANCE: Many cells in our body undergo chemotaxis, directed movement toward a chemical compound which is necessary for many biological functions including wound healing and the development of the nervous system. Chemotaxis also plays a role in disease states such as arthritis, cancer, and multiple sclerosis. Our proposed research would help us understand how cells maintain cell polarity and directed cell movement when they migrate directionally toward a directional cue.